Microbial DNA Extraction Research Articles

Microbial DNA extraction method for avian feces and preen oil from diverse species.

As DNA sequencing technology continues to rapidly improve, studies investigating the microbial communities of host organisms (i.e., microbiota) are becoming not only more popular but also more financially accessible. Across many taxa, microbiomes can have important impacts on organismal health and fitness. To evaluate the microbial community composition of a particular microbiome, microbial DNA must be successfully extracted. Fecal samples are often easy to collect and are a good source of gut microbial DNA. Additionally, interest in the avian preen gland microbiome is rapidly growing, due to the importance of preen oil for many aspects of avian life. Microbial DNA extractions from avian fecal and preen oil samples present multiple challenges, however. Here, we describe a modified PrepMan Ultra Sample Preparation Reagent microbial DNA extraction method that is less expensive than other commonly used methodologies and is highly effective for both fecal and preen oil samples collected from a broad range of avian species. We expect our method will facilitate microbial DNA extractions from multiple avian microbiome reservoirs, which have previously proved difficult and expensive. Our method therefore increases the feasibility of future studies of avian host microbiomes.

Microbial Diversity Assessment in Soil from Dumping Sites: Isolation, DNA Extraction and Electrophoretic Analysis

This study aims to investigate the microbial diversity present in soil samples collected from various dumping sites, which are known for their complex and heterogeneous waste environments. The primary objective is to isolate and characterize the microbial communities inhabiting these soils, providing insights into their composition and potential ecological roles. Soil samples were collected by digging 5 cm deep at multiple dumping locations to ensure a representative sampling of the microbial populations. The collected soil samples underwent a serial dilution process, extending up to 10^-9 and 10^-10 dilutions, to facilitate the isolation of individual microbial colonies. These diluted samples were then inoculated onto Standard Potato Dextrose Agar (PDA) media, a nutrient-rich medium conducive to microbial growth. Following an incubation period, distinct bacterial colonies were observed and isolated for further analysis. DNA was extracted from the isolated bacterial colonies using a standard DNA extraction protocol. The quality and purity of the extracted DNA were assessed through gel electrophoresis, a technique that separates DNA fragments based on their size. Remarkably, the electrophoretic analysis revealed clear and distinct DNA bands along with RNA bands, indicating successful extraction of high-quality microbial DNA. Notably, this was achieved without the use of proteinase treatment, which is typically employed to remove protein contaminants from DNA samples. The results of this study underscore the robustness of the methodologies employed in isolating and characterizing microbial DNA from soil samples collected at dumping sites. The presence of distinct DNA and RNA bands highlights the effectiveness of the DNA extraction process, even in the absence of proteinase treatment. These findings provide valuable insights into the microbial diversity within dumping site soils, suggesting a rich and varied microbial community that may play significant roles in soil health and waste decomposition.

Feed Restriction in Angus Steers Impacts Ruminal Bacteria, Its Metabolites, and Causes Epithelial Inflammation

We identified alterations in the ruminal microbiome, metabolome, and epithelial inflammatory response due to moderate feed restriction (FR). Ruminal digesta and epithelial biopsies from seven ruminally cannulated Angus steers were initially collected during ad libitum access to feed (PRE). After a 10 day recovery, steers underwent a 3-day FR period (FRP) at 25% intake of PRE followed by a 15 day recovery (POST) phase with ad libitum access to feed. At the end of FRP and POST, ruminal digesta and epithelial biopsies were collected again for microbial DNA and tissue RNA extraction. RT-qPCR was applied for relative microbial abundance and RNA extraction. Metabolite profiling of digesta was performed via GC-MS. The abundance of Succinivibrio dextrinosolvens, Streptococcus bovis, and Bifidobacteria spp. (N124) was higher (p < 0.05) during FRP than PRE and POST, while Lactobacillus spp. (C25), Escherichia coli (EC42405), Fibrobacter succinogenes, and Megaspheara elsdenii abundances were lower in FRP than PRE (p < 0.05). The TNF and TLR2 mRNA abundance was greater in FRP than PRE (p < 0.05). Among 15 detected amino acids, glutamine, isoleucine, lysine, phenylalanine, threonine, and valine were lower (p < 0.05) in FRP than PRE. Metabolite pathway analysis revealed alterations in amino acid, fatty acid, vitamin, and energy metabolism during FRP (p < 0.05). The mRNA of the proinflammatory genes TNF and TLR2 in the epithelium peaked (p < 0.05) at FRP and remained higher at POST. Results indicated that a short FR influenced ruminal bacteria, reduced concentrations of most metabolites, and triggered an inflammatory response.

A Comparison of Different Protocols for the Extraction of Microbial DNA Inhabiting Synthetic Mars Simulant Soil.

Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals, sulfates, and metal oxides. This unique soil matrix poses a major challenge for extracting microbial DNA. In particular, mineral adsorption and the generation of destructive hydroxyl radicals through cationic redox cycling may interfere with DNA extraction. This study evaluated different protocols for extracting microbial DNA from Mars Global Simulant (MGS-1), a Mars simulant soil. Two commercial kits were tested: the FastDNA SPIN Kit for soil ("MP kit") and the DNeasy PowerSoil Pro Kit ("PowerSoil kit"). MGS-1 was incubated with living soil for five weeks, and DNA was extracted from aliquots using the kits. After extraction, the DNA was quantified with a NanoDrop spectrophotometer and used as the template for 16S rRNA gene amplicon sequencing and qPCR. The MP kit was the most efficient, yielding approximately four times more DNA than the PowerSoil kit. DNA extracted using the MP kit with 0.5 g soil resulted in 28,642-37,805 16S rRNA gene sequence reads and 30,380-42,070 16S rRNA gene copies, whereas the 16S rRNA gene could not be amplified from DNA extracted using the PowerSoil kit. We suggest that the FastDNA SPIN Kit is the best option for studying microbial communities in Mars simulant soils.

EXPLORING THE MICRO AND MACRO TERROIR OF FETEASCĂ NEAGRĂ WINE FROM MOLDOVA

This study delves into the intricate realm of Moldovan winemaking, focusing on the iconic Fetească Neagră grape variety. The research highlights the importance of indigenous grape varieties in shaping a unique wine identity for the region. Drawing on the concept of terroir, the study investigates the microbial terroir specific to three designated geographical regions in Moldova during the 2022 vintage. The research employs a multifaceted approach, combining climate data analysis, mini vinification experiments, microbial DNA extraction, and sensory evaluation techniques. Results reveal significant variations in climatic conditions and microbial diversity across the three regions, influencing the sensory characteristics of the resulting wines. On vintage 2022 the Codru is temperature， and the Stefan-Voda and Vadul lui Traian are warm climates. All the 3 regions are moderately dry. The Stefan Voda is 14.0%， and manifests the highest ethanol content among the regions, alongside the lowest pH level. The sensory evaluations show Stefan Voda evincing heightened creaminess, Codru showcasing prominent herbal nuances, and Vadul lui Traian displaying elevated scores in berry, cherry, violet, and black pepper descriptors. Through meticulous analysis, the study underscores the dynamic interplay between environmental factors, microbial communities, and wine attributes, shedding light on the complex tapestry of Moldovan winemaking. Ultimately, this research contributes to a deeper understanding of terroir-driven wine production and emphasizes the importance of preserving indigenous grape varieties to uphold the distinctiveness of Moldovan wines on the global stage.

DNA extraction using FTA Elute cards for detection and enumeration of fecal indicator bacteria in the Chao Phraya River, Thailand

Utilizing molecular methods for monitoring and determining water quality in developing countries can be problematic. Effective extraction and storage of DNA from environmental samples is especially difficult due to lack of access to sufficient laboratory facilities for molecular work. Use of a commercial product for the extraction of DNA, as well as its application in determining the microbial quality of surface waters, was evaluated and compared. Surface water samples collected from the Chao Phraya River in Thailand were processed to enumerate Escherichia coli. Quantification was done through culture methods (Colilert Quanti-tray) and quantitative real-time PCR (SYBR green and Taqman assays). Processing of surface water samples and culture enumeration of E. coli was done in Thailand, while DNA extraction and enumeration using real-time PCR was done in Korea. E. coli MPN counts were mean log 2.89/100ml. Quantitative real-time PCR assays counts ranged from 2.38 to 3.59 mean log CFU/100 ml. Taqman PCR assays had no significant difference in mean log counts compared to conventional culture methods. However mean log counts of SYBR green PCR assays were significantly higher than counts obtained by culture methods and Taqman PCR assays (P<0.05). Results demonstrated FTA Elute cards can be employed for the extraction of microbial DNA from surface waters and have potential use in detection of fecal indicator bacteria from environmental water samples.

Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

Search for carbapenem-resistant bacteria and carbapenem resistance genes along swine food chains in Central Italy.

The presence of carbapenem-resistant bacteria and carbapenem resistance genes (CRGs) in livestock is increasing. To evaluate the presence of carbapenemase-producing Enterobacteriaceae (CPE) and the main CRGs along swine food chains of the Marche Region (Central Italy), samples of faeces, feed, and animal-food derived products were collected from seven small/medium, medium, and large-scale pig farms. A total of 191 samples were analysed using a culture-dependent method, with the aim of isolating CPE. Isolates were analysed for their resistance to carbapenems using a modified Hodge test and the microdilution method for the minimum inhibitory concentration (MIC) determination. Moreover, the extraction of microbial DNA from each sample was performed to directly detect selected CRGs via qPCR. Among the 164 presumptive resistant isolates, only one strain from a liver sample, identified as Aeromonas veronii, had an ertapenem MIC of 256 μg/mL and carried a carbapenemase- (cphA) and a β-lactamase- (blaOXA-12) encoding genes. A low incidence of CRGs was found; only nine and four faecal samples tested positive for blaNDM-1 and blaOXA-48, respectively. Overall, the importance of monitoring CPE and CRGs in livestock and their food chains should be stressed to control all potential non-human CPE and CRGs reservoirs and to determine safety levels for human health.

Direct identification of pathogens via microbial cellular DNA in whole blood by MeltArray.

Rapid identification of pathogens is critical for early and appropriate treatment of bloodstream infections. The various culture-independent assays that have been developed often have long turnaround times, low sensitivity and narrow pathogen coverage. Here, we propose a new multiplex PCR assay, MeltArray, which uses intact microbial cells as the source of genomic DNA (gDNA). The successive steps of the MeltArray assay, including selective lysis of human cells, microbial cell sedimentation, microbial cellular DNA extraction, target-specific pre-amplification and multiplex PCR detection, allowed the detection of 35 major bloodstream infectious pathogens in whole blood within 5.5 h. The limits of detection varied depending on the pathogen and ranged from 1 to 5 CFU/mL. Of 443 blood culture samples, including 373 positive blood culture samples and 70 negative blood culture samples, the MeltArray assay showed a sensitivity of 93.8% (350/373, 95% confidence interval [CI] = 90.7%-96.0%), specificity of 98.6% (69/70, 95% CI = 91.2%-99.9%), positive predictive value of 99.7% (95% CI = 98.1%-99.9%), and negative predictive value of 75.0% (95% CI = 64.7%-83.2%). The MeltArray detection results of 16 samples differed from MALDI-TOF and were confirmed by Sanger sequencing. Further testing of 110 whole blood samples from patients with suspected bloodstream infections using blood culture results revealed that the MeltArray assay had a clinical sensitivity of 100% (9/9, 95% CI = 62.8%-100.0%), clinical specificity of 74.5% (70/94, 95% CI = 64.2%-82.7%), positive predictive value of 27.3% (95% CI = 13.9%-45.8%), and negative predictive value of 100.0% (95% CI = 93.5%-100.0%). Compared with metagenomic next-generation sequencing, the MeltArray assay displayed a positive agreement of 85.7% (6/7, 95% CI = 42.0%-99.2%) and negative agreement of 100.0% (4/4, 95% CI = 39.6%-100.0%). We conclude that the MeltArray assay can be used as a rapid and reliable tool for direct identification of pathogens in bloodstream infections.

Expanded Multiplexing on Sensor-Constrained Microfluidic Partitioning Systems.

Microfluidics can split samples into thousands or millions of partitions, such as droplets or nanowells. Partitions capture analytes according to a Poisson distribution, and in diagnostics, the analyte concentration is commonly inferred with a closed-form solution via maximum likelihood estimation (MLE). Here, we present a new scalable approach to multiplexing analytes. We generalize MLE with microfluidic partitioning and extend our previously developed Sparse Poisson Recovery (SPoRe) inference algorithm. We also present the first in vitro demonstration of SPoRe with droplet digital PCR (ddPCR) toward infection diagnostics. Digital PCR is intrinsically highly sensitive, and SPoRe helps expand its multiplexing capacity by circumventing its channel limitations. We broadly amplify bacteria with 16S ddPCR and assign barcodes to nine pathogen genera by using five nonspecific probes. Given our two-channel ddPCR system, we measured two probes at a time in multiple groups of droplets. Although individual droplets are ambiguous in their bacterial contents, we recover the concentrations of bacteria in the sample from the pooled data. We achieve stable quantification down to approximately 200 total copies of the 16S gene per sample, enabling a suite of clinical applications given a robust upstream microbial DNA extraction procedure. We develop a new theory that generalizes the application of this framework to many realistic sensing modalities, and we prove scaling rules for system design to achieve further expanded multiplexing. The core principles demonstrated here could impact many biosensing applications with microfluidic partitioning.

Anionic nanocellulose as competing agent in microbial DNA extraction from mine process samples

Microorganisms in flotation and minerals processing may significantly affect the grade and yield of metal concentrates. However, studying the phenomena requires working techniques to detach microorganisms and their DNA from mineral particles to which they strongly adhere. We developed a new method utilizing the competitive properties of anionic nanocellulose to block sorption of DNA to and detach microbial cells from mineral particles from ore processing. In general, up to one ng DNA mL−1 sample was obtained with the custom anionic nanocellulose method (CM) compared to DNA amounts below the Qubit assay's detection limit for extractions with a commercial kit (KIT). Similarly, 0.5–4 orders of magnitude more bacterial 16S and fungal 5.8S rRNA gene copies were detected by qPCR from CM treated samples compared to KIT extractions. A clear difference in the detected microbial community structure between CM and KIT extracted samples was also observed. Commercial kits optimized for mineral soils are easy to use and time efficient but may miss a considerable part of the microbial communities. A competing agent such as anionic nanocellulose may decrease the interaction between microorganisms or their DNA and minerals and provide a comprehensive view into the microbial communities in mineral processing environments.

Metagenomic analysis reveals a dynamic microbiome with diversified adaptive functions that respond to ovulation regulation in the mouse endometrium

Understanding the microflora inhabiting the reproductive tract is important for a better understanding of female physiology and reproductive health. The endometrial fluid from mice in three reproductive stages (A: Unproductive mice; B: Postovulatory mice; C: Postpartum mice) was extracted for microbial DNA extraction and sequencing. Phenotypic and functional analyses of endometrial microbial enrichment was undertaken using LefSe. The results showed 95 genera and 134 species of microorganisms in the uteri of mice. There were differentially distributed genera, among which Lactobacillus, Enterococcus, and Streptococcus were more abundant in the endometrial fluid of mice in the unproductive group. That of mice in the postovulatory group was colonized with Salmonella enterica and Campylobacter and was mainly enriched in metabolic pathways and steroid biosynthesis. The presence of Chlamydia, Enterococcus, Pseudomonadales, Acinetobacter, and Clostridium in the endometrial fluid of postpartum mice, in addition to the enrichment of the endocrine system and the Apelin and FoxO signaling pathways, resulted in a higher number of pathogenic pathways than in the other two groups. The results showed that the microbial diversity characteristics in the endometrium of mice in different reproductive states differed and that they could be involved in the regulation of animal reproduction through metabolic pathways and steroid biosynthesis, suggesting that reproductive diseases induced by microbial diversity alterations in the regulation of animal reproduction cannot be ignored.

Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics.

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

16S rRNA gene-based microbiota profiles from diverse avian faeces are largely independent of DNA preservation and extraction method.

The avian gut microbiota has been the subject of considerable recent attention, with potential implications for diverse fields such as the poultry industry, microbial ecology, and conservation. Faecal microbiotas are frequently used as a non-invasive proxy for the gut microbiota, however the extraction of high-quality microbial DNA from avian faeces has often proven challenging. Here we aimed to evaluate the performance of two DNA preservation methods (95% ethanol and RNAlater) and five extraction approaches (IndiSpin Pathogen Kit, QIAamp PowerFecal Pro DNA Kit, MicroGEM PrepGEM Bacteria Kit, ZymoBIOMICS DNA Miniprep Kit, and an in-house phase separation-based method) for studying the avian gut microbiota. Systematic testing of the efficacy of these approaches on faecal samples from an initial three avian species (chicken, ostrich, and the flightless parrot kākāpō) revealed substantial differences in the quality, quantity and integrity of extracted DNA, but negligible influence of applied method on 16S rRNA gene-based microbiota profiles. Subsequent testing with a selected combination of preservation and extraction method on 10 further phylogenetically and ecologically diverse avian species reiterated the efficacy of the chosen approach, with bacterial community structure clustering strongly by technical replicates for a given avian species. Our finding that marked differences in extraction efficacy do not appear to influence 16S rRNA gene-based bacterial community profiles provides an important foundation for ongoing research on the avian gut microbiota.

Variability in chewing, ruminal fermentation, digestibility and bacterial communities between subacute ruminal acidosis-susceptible and acidosis-tolerant sheep.

Sorting behaviour is a common phenomenon observed in ruminants when they are provided with a total mixed ration, which contributes to variations in the severity of subacute ruminal acidosis (SARA). Pelleted total mixed ration (PTMR) reduces sorting, but high-grain content increases acidosis risk. However, whether the variability in the severity of SARA exists in sheep fed the same high-grain PTMR is less understood. This study aimed to investigate SARA variability among individual sheep offered a high-grain PTMR, considering chewing activity, ruminal fermentation, bacterial communities and nutrient digestibility. Twenty ruminally cannulated male Hu sheep were individually housed in cages and fed a PTMR comprising 80% concentrate mix and 20% roughage. A 14-day adaptation period to the diet and facilities was provided before a 10-day sample collection period. Continuous monitoring of ruminal pH was conducted for 48h, during which time chewing activity was also recorded. Ruminal fluid samples were collected for analysis of volatile fatty acid and microbial DNA extraction. Faecal samples were collected to measure nutrient digestibility. Based on their acidosis index, the sheep were classified into two groups: SARA-susceptible group (n=6) and SARA-tolerant group (n=6). The SARA-susceptible sheep exhibited a lower ruminal mean pH and minimum pH than the SARA-tolerant sheep (P<0.05). Additionally, the SARA-susceptible group increased the acidosis index, duration and areas of pH below 5.8 and 5.6 compared to the SARA-tolerant group (P<0.05). The SARA-susceptible group also exhibited a longer ruminating time than the SARA-tolerant group (P<0.05). The SARA-susceptible group exhibited a tendency to increase the relative abundance of Firmicutes (P=0.089), while simultaneously decreasing the copy number of Fibrobacter succinogenes in the rumen, as well as the digestibility of NDF and ADF compared to the SARA-tolerant group (P<0.05). The acidosis index was found to be positively correlated with ruminating time (min/kg DM intake (DMI)) and total chewing time (min/kg DMI), but negatively correlated with the copy number of Fibrobacter succinogenes and Ruminococcus albus in the rumen. These findings indicate that there exists variability in the SARA severity among sheep when fed a high-grain PTMR, as evidenced by varied chewing activity, bacterial communities and nutrient digestibility. Ruminating time, total chewing time per kilogram of DMI as well as the copy number of Fibrobacter succinogenes and Ruminococcus albus in the rumen hold potential as indicators for assessing the severity of SARA.

High Mobility Group Box 1: A Predictive Biomarker of Aberrant Humoral Responses Implicated in Post-Acute Sequelae of SARS-CoV-2 Infection

Abstract Postacute sequelae of SARS-CoV-2 infection (PASC) is the next public health concern of the pervasive COVID-19 pandemic. Molecular pathophysiology of PASC is undetermined, therefore this study evaluated pathways implicated in humoral responses associated with symptom resolution to identify predictive biomarkers of PASC. Individuals recovered from acute infection were recruited into a 6-week study. Initial health questionnaires and proof of previous SARS-CoV-2 infection were collected prior to study enrollment. Body mass indices were calculated with participant height and weight measurements. Blood samples were collected and isolated for plasma and PBMCs to evaluate a panel of immunological biomarkers and cellular profiling, respectively. A singular stool sample was collected for microbial DNA extraction for metagenomic sequencing. Data was integrated with IPA analysis to establish modulated pathways in PASC. Individuals suffering from symptoms of PASC showed evidence of poor immune responses with diminished rates of symptom resolution. Particularly prevalent in obese individuals, PASC associated with attenuated antibody responses and inability to maintain viral inhibition capacities, partially explained by elevated inflammation and inflammatory cellular landscapes. Individuals with worse humoral responses tended to display characteristics of a compromised gut microbiome, suggesting dysregulation of the immunoepigenetic-microbiome axis. Circulating high mobility group box 1 was shown to be a central mediator of this axis, with elevated levels conducive of poor adaptive immunity. Additionally, mediation analysis revealed baseline levels were predictive of viral inhibition at the time of final follow-up. This study was supported in part by funding provided by Hawaii Community Foundation under award 20HCF-101573. The comments expressed in this report are the sole responsibility of the authors and do not represent the official view of the Hawaii Community Foundation.

Impact of Molasses on Ruminal Volatile Fatty Acid Production and Microbiota Composition In Vitro.

The aim of this study was to assess if molasses could modify VFA production and the rumen microbial community in vitro. Three beet (treatment Beet) and three cane (treatment Cane) molasses preparations were randomly selected from a variety of samples collected worldwide and incubated in vitro with rumen fluid along with a control sample (treatment CTR, in which no molasses was used). Flasks for VFA analysis were sampled at 0, 1, 2, 3, 4, 6, 8, and 24 h of each incubation. For microbiota analysis, samples from each fermentation flask after 12 and 24 h were subjected to microbial DNA extraction and V3-V4 16S rRNA gene sequencing on an Illumina MiSeq platform. Total net VFA production was higher in the beet and cane preparations than in the control (CTR) group at 24 h (33 mmol/L, 34 mmol/L, and 24.8 mmol/L, respectively), and the composition of VFAs was affected by the inclusion of molasses: acetic acid increased in the CTR group (73.5 mol%), while propionic acid increased in the beet and cane molasses (19.6 mol% and 18.6 mol%, respectively), and butyric acid increased, especially in the cane group (23.2 mol%). Molasses even influenced the composition of the rumen microbiota, and particularly the relative abundance of the most dominant family in the rumen, Prevotellaceae, which decreased compared to CTR (37.13%, 28.88%, and 49.6%, respectively). In contrast, Streptococcaceae (19.62% and 28.10% in molasses compared to 6.23% in CTR), Veillonellaceae (6.48% and 8.67% in molasses compared to 4.54% in CTR), and Fibrobacteraceae (0.90% and 0.88% in molasses compared to 0.62% in CTR) increased in the beet and cane groups compared to the CTR group. Another important finding is the lower proportion of Methanobacteriaceae following the addition of molasses compared to CTR (0.26%, 0.28%, and 0.43%, respectively). This study showed the impact of molasses in influencing VFA production and composition as a result of a modified rumen microbial composition.

Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

Plant Products as Biocides for Conservation of Cultural Asset Sustainable for Human and Environmental Health

In the last decades plants products are increasingly used also in defining innovative procedures for sustainable conservation of cultural heritage. Particularly, essential oils or hydro-alcoholic extracts have been fruitful applied to contrast microbial colonization on organic and inorganic artworks or insect infestation (Anobidae) having regard to the repellent action.&#x0D; In this paper, extracts from Asteraceae and Lamiaceae plant families are utilized to counteract widespread microbial colonization (bacteria, cyanobacteria, fungi) due to their antimicrobial activity. In order to define the adequate concentration correlating it to microbial species detected, the antimicrobial activity of natural products is preliminary in vitro assayed by Agar Disc and Well Plate Diffusion methods. Moreover, an integrated approach based on morphological analysis (optical, scanning and confocal electron microscopy), in vitro cultures (Nutrient or Sabouraud agar) and molecular investigation (genomic microbial DNA extraction, sequencing and sequence analysis) are routinely performed for the identification of largest number of microbial taxa.&#x0D; Besides, to enhance the antimicrobial activity different protocols have been performed, such as for Thymus vulgaris products directly applied on wooden sculpture surface as hydro-alcoholic extract, followed by exposure, in a dedicated chamber, to the volatile compound of the corresponding Essential Oil. In other case studies, the exposure to volatile compounds of Crithmum maritmum, Inula critmoides, Thymus vulgaris and Origanum vulgare essential oils was performed under controlled vacuum conditions, comparing the effects to environmental condition exposure. These studies confirm the possible use of plant extracts in replacing synthetic chemical biocides, in full respect of human health and environment sustainability.